The present invention relates generally to therapeutic compositions and treatment methods utilizing bactericidal/permeability-increasing protein (BPI) protein products for the treatment of thrombotic disorders.
The coagulation, or blood clotting process is involved both in normal hemostasis, in which the clot stops blood loss from a damaged blood vessel, and in abnormal thrombosis, in which the clot blocks circulation through a blood vessel. During normal hemostasis, the platelets adhere to the injured blood vessel and aggregate to form the primary hemostatic plug. The platelets then stimulate local activation of plasma coagulation factors, leading to generation of a fibrin clot that reinforces the platelet aggregate. Later, as wound healing occurs, the platelet aggregate and fibrin clot are degraded by specifically activated proteinases. During the pathological process of thrombosis, the same mechanisms create a platelet/fibrin clot that occludes a blood vessel. Arterial thrombosis may produce ischemic necrosis of the tissue supplied by the artery, e.g., myocardial infarction due to thrombosis of a coronary artery, or stroke due to thrombosis of a cerebral artery. Venous thrombosis may cause the tissues drained by the vein to become edematous and inflamed, and thrombosis of a deep vein may result in a pulmonary embolism.
An increased tendency toward thrombosis accompanies surgery, trauma, many inflammatory disorders, malignancy, pregnancy, obesity, vascular disorders and prolonged immobilization. Inherited thrombotic tendencies, which are much rarer, are being increasingly recognized and include deficiencies of the protein C-protein S system, deficiencies of antithrombin III (ATIII), dysfibrinogenemias, and other disorders of the fibrinolytic system. The evaluation of hypercoagulable risk involves checking for a family history of thromboembolism, and for other systemic predisposing diseases or conditions that favor localized vascular stasis (such as prolonged immobilization, pregnancy, or malignancy) and evaluating possible laboratory abnormalities, such as thrombocytosis, elevated blood or plasma viscosity, and elevated plasma levels of coagulation factors or fibrin degradation products. Levels of ATIII, protein C, or protein S levels, may also be measured, although hypercoagulability due to such abnormalities is uncommon compared to factors such as stasis or localized injury.
Severe derangements of the coagulation process are seen in disseminated intravascular coagulation (DIC), a syndrome characterized by the slow formation of fibrin microthrombi in the microcirculation and the development of concomitant fibrinolysis. The net result of these processes is the consumption of platelets and clotting factors in the thrombotic process, and the proteolytic digestion of several clotting factors by the fibrinolytic process, leading to decreased coagulability of the patient's blood. DIC never occurs as a primary disorder; it is always secondary to another disorder. These primary disorders fall into three general categories: (1) release of procoagulant substances into the blood, as may occur in amniotic fluid embolism, abruptio placentae, certain snake bites, and various malignancies, (2) contact of blood with an injured or abnormal surface, as may occur in extensive burns, infections, heat stroke, organ grafts, and during extracorporal circulation, and (3) generation of procoagulant-active substances within the blood, as may occur if red or white blood cell or platelet membranes become damaged and release thromboplastic substances, e.g., during leukemia treatment, hemolytic transfusion reactions and microangiopathic hemolytic anemia. Bacterial endotoxins on, associated with or released from gram-negative bacteria also have thromboplastin-like properties that initiate clotting.
Intravascular clotting occurs most frequently with shock, sepsis, cancer, obstetric complications, burns, and liver disease. There are no specific symptoms or signs unique to DIC. Bleeding, however, is much more evident than thrombosis. The rate and extent of clotting factor activation and consumption, the concentration of naturally occurring inhibitors, and the level of fibrinolytic activity determine the severity of the bleeding tendency. In some patients there is no clinical evidence of bleeding or thrombosis, and the syndrome becomes apparent only as a consequence of abnormal blood coagulation tests. Many patients develop only a few petechiae and ecchymotic areas and bleed a little more than usual from venipuncture sites. More pronounced forms of diffuse intravascular clotting may become evident as a result of severe gastrointestinal hemorrhage or genitourinary bleeding. In some instances bleeding may cause death. Hemorrhage caused by the DIC syndrome can be especially life threatening in association with obstetric complications or in conjunction with surgery.
The endpoint of the coagulation process is the generation of a powerful serine protease, thrombin, which cleaves the soluble plasma protein fibrinogen so that an insoluble meshwork of fibrin strands develops, enmeshing red cells and platelets to form a stable clot. This coagulation process can be triggered by injury to the blood vessels and involves the rapid, highly controlled interaction of more than 20 different coagulation factors and other proteins to amplify the initial activation of a few molecules to an appropriately sized, fully developed clot. Most of the coagulation proteins are serine proteases that show a high degree of homology (Factors II, VII, IX, and X); others are cofactors without enzyme activity (Factors V and VIII). These proteins circulate as inactive zymogens in amounts far greater than are required for blood clotting. Both the injured vessel wall and platelet aggregates provide specialized surfaces that localize and catalyze the coagulation reactions.
The coagulation cascade can be initiated via two different activation pathways: the intrinsic pathway, involving contact with injured tissue or other surfaces, and the extrinsic pathway, involving tissue factor expressed on injured or inflamed tissue. Both pathways converge into a common pathway when Factor X is activated at the platelet surface. [See, e.g., Cecil's Essentials of Medicine, 3rd ed., WB Saunders Co., Pennsylvania (1983); Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, N.Y. (1996).] The intrinsic pathway begins when Factor XII is activated to XIIa by contact with the altered or injured blood vessel surface or with another negatively charged surface, such as a glass tube. Cofactors or promoters of Factor XII activation include prekallikrein, high molecular weight kininogen, and Factor XI. These proteins form a surface-localized complex which optimally activates Factor XII. The activated Factor XIIa then converts the complex-bound Factor XI to its active form, XIa, and also converts prekallikrein to its active form, kallikrein, which then cleaves high molecular weight kininogen to form bradykinin. In turn, Factor XIa requires calcium ions (Ca.sup.2+) to activate Factor IX to IXa. Factor XIa may also activate Factor VII (in the extrinsic pathway) as well. Activated Factor XIa also cleaves plasminogen to form plasmin, which is the main protease involved in the fibrinolytic mechanisms that restrain blood clotting. In the presence of Ca.sup.2+ and phospholipid, Factor IXa activates Factor X to Xa, which is the first step in the common pathway. Factor X activation usually takes place at the plasma membrane of stimulated platelets but also may occur on the vascular endothelium.
In the extrinsic pathway, the release of tissue factor from injured tissues directly activates Factor VII to VIIa. Tissue factor is present in activated endothelium and monocytes as well as in brain, vascular adventitia, skin, and mucosa. Factor VIIa then activates Factor X to Xa in the presence of Ca.sup.2+. In addition, the tissue factor, Factor VII, and Ca.sup.2+ form a complex that can activate Factor IX (in the intrinsic pathway).
The activated Factor Xa (the first step in the common pathway) then activates prothrombin (Factor II) to generate the protease thrombin. Assembly of the plasma prothrombinase complex on the surface of activated platelets in the presence of Factor V, another cofactor, enhances the efficiency of prothrombin activation to thrombin on the platelet surface. Thrombin cleaves fibrinogen, which is a large, asymmetric, soluble protein with a molecular weight of about 340 kilodaltons consisting of three pairs of polypeptide chains: A.alpha., B.beta., and .gamma.. Thrombin first removes small peptides from the A.alpha. chain of fibrinogen to form Fibrin I, which polymerizes end to end; further thrombin cleavage of small peptides from the B.beta. chain leads to formation of Fibrin II molecules, which polymerize side to side and are then cross-linked via the .gamma. subunits by the plasma glutaminase (Factor XIII) to form an insoluble fibrin clot.
Thrombin has multiple critical actions during coagulation in addition to the cleavage of fibrinogen to fibrin. It activates platelets, exposing their procoagulant activity (e.g., binding sites for the prothrombinase complex) and induces the release of platelet-aggregating substances such as thromboxane, Ca.sup.2+, ADP, von Willebrand factor, fibronectin, and thrombospondin. Thrombin cleaves Factors VIII and Va, thus augmenting the coagulation cascade, and also cleaves plasma glutaminase, the enzyme which cross-links fibrin and stabilizes the fibrin clot. Thrombin acts on the endothelium by binding to the surface protein thrombomodulin to activate protein C, which is a potent inactivator of Factors Va and VIII and also stimulates fibrinolysis. Thrombin also causes endothelial cell contraction. Conversely, endothelium can bind and inactivate thrombin, and in some cases can generate the vasodilatory substance prostacyclin in response to thrombin. Thus, thrombin activation contributes to the limitation as well as the initiation of clotting.
There are two commonly used tests for measuring the coagulability of blood: the activated partial thromboplastin time (APTT or PTT) and the prothrombin time (PT). Blood generally clots in vitro in four to eight minutes when placed in a glass tube. Clotting is prevented if a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or citrate is added to bind Ca.sup.2+. Recalcified plasma, i.e., plasma in which Ca.sup.2+ has been replenished, clots in two to four minutes. The clotting time after recalcification is shortened to 26 to 33 seconds by the addition of negatively charged phospholipids and a particulate substance such as kaolin (aluminum silicate); this post-recalcification clotting time is the APTT. Alternatively, recalcified plasma will clot in 12 to 14 seconds after addition of "thromboplastin," a saline extract of brain that contains tissue factor and phospholipids; this post-recalcification clotting time is the PT.
An individual with a prolonged APTT and a normal PT is considered to have a defect in the intrinsic coagulation pathway, because all of the components of the APTT test (except kaolin) are intrinsic to the plasma. A patient with a prolonged PT and a normal APTT has a defect in the extrinsic coagulation pathway, since thromboplastin is extrinsic to the plasma. Prolongation of both the APTT and the PT suggests a defect in a common pathway.
Whereas the blood coagulation pathways involve a series of enzymatic activations of serine protease zymogens, downregulation of blood clotting is influenced by a variety of natural anticoagulant mechanisms, including antithrombin III (ATIII), the protein C-protein S system, and fibrinolysis. Normal vascular endothelium promotes the activation of these anticoagulant mechanisms by acting as a source of heparin-like substances that enhance ATIII activation, a source of thrombomodulin, a cofactor in protein C activation, and a source of the tissue plasminogen activators that initiate fibrinolysis.
The anticoagulant ATIII is a plasma protease inhibitor that is specific for plasmin, the enzyme that dissolves clots. ATIII also binds all the serine protease procoagulant proteins (Factor Xa as well as thrombin). Complexes of ATIII and protease are rapidly cleared by the liver and the reticuloendothelial system. The activity of ATIII is enhanced by heparin or heparin-like substances. Other enzymes that play a role in limiting the coagulation process include the nonspecific plasma protease inhibitors .alpha..sub.1 -antitrypsin, .alpha..sub.2 -plasmin inhibitor, and .alpha..sub.2 -macroglobulin, which rapidly inactivate any circulating serine proteases including thrombin and plasmin.
The final stage of the coagulation process is fibrinolysis, or clot dissolution. The endpoint of the fibrinolytic system is the generation of the enzyme plasmin, which dissolves intravascular clots by digesting fibrin. Fibrinolysis is initiated during clotting by the action of thrombin. When complexed to thrombomodulin in the endothelium, thrombin activates protein C, which initiates the release of tissue plasminogen activator (tPA) from the blood vessel wall. Protein C, together with its cofactor protein S, also inactivates Factors Va and VIIIa, thus dampening the coagulation cascade. The tPA then cleaves a circulating proenzyme, plasminogen, to form the active protease, plasmin, which digests fibrin. Plasmin is a relatively nonspecific protease; it not only digests fibrin clots but also digests other plasma proteins, including several coagulation factors.
The fibrinolytic system is regulated in a manner so that unwanted fibrin thrombi are removed, while fibrin in wounds persists to maintain hemostasis. The tPA is released from endothelial cells in response to various signals, including stasis produced by occlusion of the blood vessel. This released tPA exerts little effect on circulating plasminogen because tPA is rapidly cleared from blood or inhibited by circulating inhibitors, plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2. Both plasminogen and its activator tPA bind to fibrin. The activity of tPA is actually enhanced by this binding to fibrin, so that the generation of plasmin is localized to the vicinity of the blood clot. In addition, fibrin-bound plasmin is protected from inhibition.
Four main types of therapies are used to prevent or treat thrombosis: antiplatelet agents, anticoagulant agents (heparin), vitamin K antagonists (coumarin derivatives) and thrombolytic agents. Each type of agent interferes with clotting at a different site in the coagulation pathway [See, generally, Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, N.Y. (1996).] Dipyridamole is another agent sometimes used to prevent or treat thrombosis; it is a vasodilator that, in combination with warfarin (a coumarin derivative), inhibits embolization from prosthetic heart valves and, in combination with aspirin, reduces thrombosis in patients with thrombotic disorders.
The antiplatelet agents include aspirin and other non-steroidal anti-inflammatory agents such as ibuprofen, which are all administered orally. Aspirin acts by irreversibly inhibiting platelet cyclooxygenase and thus blocking production of thromboxane A.sub.2, an inducer of platelet aggregation and potent vasoconstrictor. In general, antiplatelet agents are used as prophylaxis against arterial thrombosis, because platelets are more important in initiating arterial than venous thrombi. Antiplatelet therapy also reduces the risk of occlusion of saphenous vein bypass grafts.
The anticoagulant agents include heparin and its derivatives, which act by accelerating the activities of ATIII in inhibiting thrombin generation and in antagonizing thrombin's action. Low molecular weight preparations of heparin such as dalteparin and enoxaparin may also be effective for anticoagulation. Heparin increases the rate of the thrombin-antithrombin reaction at least a thousandfold by serving as a catalytic template. Heparin can only be administered parenterally and has an immediate anticoagulant effect. It is used to prevent and treat arterial and venous thrombosis, as well as to keep blood fluid during extracorporeal circulation, such as with renal hemodialysis or during cardiopulmonary bypass, and to keep vascular access catheters patent. Heparin therapy is also standard in patients undergoing percutaneous transluminal coronary angioplasty.
Bleeding is the primary adverse effect of heparin. Major bleeding occurs in 1% to 33% of patients who receive various forms of heparin therapy. Purpura, ecchymoses, hematomas, gastrointestinal hemorrhage, hematuria, and retroperitoneal bleeding are regularly encountered complications of heparin therapy. Frequently bleeding is most pronounced at sites of invasive procedures. If bleeding is severe, the effects of heparin can be counteracted by giving 1 mg of protamine sulfate for each 100 units of heparin. Another side effect, thrombocytopenia, also occurs in 1% to 5% of patients receiving heparin, but subsides when heparin is discontinued.
The vitamin K antagonists (coumarin derivatives) are sometimes referred to as oral anticoagulants although they do not actually directly inhibit the coagulation cascade. These agents include 4-hydroxycoumarin, warfarin sodium, dicumarol, phenprocoumon, indan-1,3-dione, acenocoumarol, and anisindione. They interfere with the hepatic synthesis of Factors II, VII, IX, and X and proteins C and S, which are all involved in the coagulation process, and therefore have a slow onset of anticoagulant effect that spans several days. They are given orally; once the dose is established for an individual patient, they can provide a steady level of anticoagulation. Vitamin K antagonists are used for both the prevention and treatment of arterial and venous thrombosis.
Bleeding is the major adverse effect of vitamin K antagonists. Especially serious episodes involve sites where irreversible damage may result from compression of vital structures (e.g., intracranial, pericardial, nerve sheath, or spinal cord) or from massive internal blood loss that may not be diagnosed rapidly (e.g., gastrointestinal, intraperitoneal, retroperitoneal). The risk of intracerebral or subdural hematoma in patients over 50 years of age taking an oral anticoagulant over a long term may be increased ten-fold. For continued or serious bleeding, vitamin K.sub.1 (phytonadione) is an effective antidote. Since reversal of anticoagulation by vitamin K.sub.1 requires the synthesis of fully carboxylated coagulation proteins, significant improvement in hemostasis does not occur for several hours, regardless of the route of administration, and 24 hours or longer may be needed for maximal effect. Warfarin is contraindicated in women who are or may become pregnant because the drug passes through placental barrier and may cause fatal hemorrhage in the fetus. Warfarin treatment during pregnancy may also cause spontaneous abortion, still birth and birth defects.
The thrombolytic agents include tPA, streptokinase, urokinase prourokinase, anisolylated plasminogen streptokinase activation complex (APSAC), and animal salivary gland plasminogen activators, all of which act by accelerating fibrinolysis. The thrombolytic drugs are used to lyse freshly formed arterial and venous thrombi; they are not efficacious in dissolving thrombi that have been present for more than a few hours. The intravenous administration of these agents is now accepted as useful therapy in the management of deep vein thrombosis, pulmonary embolism, acute myocardial infarction, and peripheral arterial thromboembolism.
The major toxicity of all thrombolytic agents is hemorrhage, which results from two factors. Therapy with thrombolytic drugs tends to dissolve both pathological thrombi and fibrin deposits at sites of vascular injury. In addition, a systemic lytic state results from systemic formation of plasmin, which produces fibrinogenolysis and destruction of other coagulation factors. Massive fibrinolysis is initiated, and the inhibitory controls of the process are overwhelmed. The systemic loss of fibrinogen and platelet dysfunction caused by the thrombolytic agents also produces a hemorrhagic tendency. Thus, the use of thrombolytic agents is contraindicated in situations where there is active bleeding or a risk of major hemorrhage.
If heparin is used concurrently with either streptokinase or t-PA, serious hemorrhage will occur in 2% to 4% of patients. Intracranial hemorrhage is by far the most serious problem; it occurs in approximately 1% of cases, and the frequency is the same with all three thrombolytic agents. Retroperitoneal hemorrhage is also a serious complication. The frequency of hemorrhage is less when thrombolytic agents are utilized to treat myocardial infarction compared with pulmonary embolism or venous thrombosis; this difference may be due to the duration of therapy (1 to 3 hours for myocardial infarction, compared to 12 to 72 hours for pulmonary embolism and venous thrombosis).
In general, venous thrombosis and its potential for life-threatening pulmonary embolism are prevented and treated with heparin or warfarin. Low-dose subcutaneous heparin is frequently used as prophylaxis against venous thrombosis in surgical patients but is ineffective in those at highest risk, for example, after hip fracture. Warfarin reduces mortality from pulmonary embolism and can be given more safely to immobilized or post-surgical patients in low-dose or stepwise regimens. Once a venous thrombosis has developed, however, full-dose heparin treatment for 5 to 10 days overlapping with full-dose warfarin treatment for 4 to 5 days is necessary to prevent clot progression and/or pulmonary embolism. Thrombolytic agents have been used to treat pulmonary embolism and deep venous thrombosis, but their efficacy in reducing mortality remains to be established. Aspirin offers little value in treating venous thromboembolism.
For acute arterial thrombosis, thrombolytic therapy is generally the treatment of choice. The goals of thrombolytic therapy are to achieve rapid reperfusion of the thrombosed vessel and maintain patency of the vessel; these objectives are based on the premise that rapid and sustained restoration of blood flow reduces associated complications. However, multiple episodes of vessel reocclusion typically follow thrombolytic therapy. Although widely used as an adjunct to thrombolytic therapy, heparin does not accelerate thrombolysis or prevent reocclusion of the vessel. [Klement et al., Thrombosis Haemostasis, 68:64-68 (1992).] In patients with a fresh coronary thrombosis, intravenous thrombolytic therapy can permit rapid reperfusion of the thrombosed coronary artery, thus preserving cardiac function and reducing mortality, if administered within a few hours of the onset of symptoms. Thrombolytic agents can also re-establish the patency of thrombosed peripheral arteries if administered within a few hours after acute thrombosis. In some instances, e.g., for coronary artery thrombosis, the thrombolytic agent is administered locally by selective catheterization of the involved vessel. When given systemically rather than locally, a therapeutic effect is evident if the thrombin time is greater than twice normal. Such treatment should generally be followed by heparin and then oral anticoagulants to prevent further clot promulgation or recurrence. Following thrombolytic therapy and before the thrombin time has returned to its normal range, heparin is generally given to fully anticoagulate the patient for five to ten days. Warfarin may be started before the heparin is stopped, depending on whether prolonged anticoagulation will be required in the management of the patient's disorder. Aspirin is ineffective in the immediate setting, but is useful for long-term prophylaxis against arterial thrombosis. Recent studies suggest that the concurrent administration of low doses of aspirin improves the efficacy of thrombolytic therapy of myocardial infarction. Patients with symptomatic strokes are acutely anticoagulated with heparin and followed indefinitely with warfarin. Aspirin is recommended for prophylaxis of stroke in patients with cervical bruits, asymptomatic carotid stenosis, or a history of transient ischemic attacks and minor stroke.
Considerable controversy continues to surround the use of heparin in DIC. Heparin is usually reserved for fulminant, explosive forms of diffuse intravascular clotting, in which massive defibrination is accompanied by fibrinogen levels of less than 100 mg/dL and replacement therapy is not controlling the hemorrhage. In these cases, heparin is given as a continuous intravenous infusion at a rate of 10 to 15 units/kg/hour. If the patient is in immediate danger of dying from hemorrhage, 5000 to 10,000 units of heparin are given intravenously as a bolus and heparin is then continued at an infusion rate of 1000 units per hour.
Heparin can also be useful in treating unstable angina and patients undergoing elective cardioversion for atrial fibrillation of greater than 2 days duration. Warfarin and aspirin are useful for prophylaxis of cerebral embolism, particularly in patients at risk because of atrial fibrillation. More than 50% of patients with cerebral embolism have atrial fibrillation. Warfarin is also recommended for treating patients with mechanical heart valves, for whom the associated risk of embolism is 2% to 6% per patient per year despite anticoagulation, patients with rheumatic mitral valve disease, in whom the rate of associated thromboembolic complications is 1.5% to 4.7% per year, and patients with a history of thromboembolism. Aspirin is recommended for patients with mitral valve prolapse.
Anti-thrombotic agents are also used routinely to prevent the occlusion of extracorporeal devices: intravascular cannulas (heparin), vascular access shunts in hemodialysis patients (aspirin), hemodialysis machines (heparin), and cardiopulmonary bypass machines (heparin). In addition, they have been utilized in the treatment of certain renal diseases (heparin/warfarin) and small-cell lung cancer (warfarin).
BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coil affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Gray et al. amino acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat. No. 5,198,541 discloses recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI.
BPI is a strongly cationic protein. The N-terninal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of -3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD possesses essentially all the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as "rBPI.sub.23," has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms as well as endotoxin-neutralizing activity. [Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).]
The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, ltd. (1992). The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to and neutralizes lipid A, reported to be the most toxic and most biologically active component of LPS.
In addition to BPI's bactericidal and endotoxin binding/neutralizing activities, BPI has been shown to bind and neutralize heparin. Co-owned U.S. Pat. No. 5,348,942 was issued Sept. 20, 1994 with claims directed to methods of neutralizing the anticoagulant effects of heparin with BPI protein products (i.e., their procoagulant activity). There has been no suggestion or use of BPI as an anticoagulant or thrombolytic agent, nor any suggestion of its use for the prophylaxis or treatment of thrombotic disorders.
There exists a need in the art for methods and compositions capable of exerting anticoagulant or thrombolytic effects without severe adverse side effects, and methods and compositions capable of improving the therapeutic effectiveness of existing anticoagulant or thrombolytic agents, which ideally could reduce the required dosages of such existing agents.